Inhibiting furin with polybasic peptides

ABSTRACT

Small, polybasic peptides are disclosed that are effective as furin inhibitors, e.g. hexa- to nona-peptides having L-Arg or L-Lys in most positions. Removing the peptide terminating groups can improve inhibition of furin. High inhibition was seen in a series of non-amidated and non-acetylated polyarginines. The most potent inhibitor identified to date, nona-L-arginine, had a K i  against furin of 40 nM. Non-acetylated, poly-D-arginine-derived molecules are preferred furin inhibitors for therapeutic uses, such as inhibiting certain bacterial infections, viral infections, and cancers. Due to their relatively small size, these peptides should be non-immunogenic. These peptides are efficiently transported across cell membranes.

[0001] The development of this invention was funded in part by theGovernment under grant number DA05084 awarded by the National Institutesof Health. The Government has certain rights in this invention.

[0002] This invention pertains to the inhibition of furin, which can beused in inhibiting certain bacterial infections, viral infections, andcancers.

[0003] Furin, a ubiquitous serine endoprotease, has been implicated inthe activation of certain bacterial toxins and viral glycoproteins, aswell as in the metastatic progression of certain tumors. Inhibitors offurin can be useful in inhibiting bacterial infections, viralinfections, and tumors that depend on furin. While some inhibitors offurin have previously been reported, they have had high molecularweights, making them relatively expensive and potentially immunogenic;or they are toxic. To the inventors' knowledge, there have been nosmall, non-toxic nanomolar inhibitors of furin reported previously.

[0004] Furin is a calcium-dependent, membrane-bound serineendoproteinase. It is a member of the “subtilisin-like”proprotein/prohormone convertase (PC) family of enzymes. The PC familyof hormones includes those known as furin; PACE-4; PC2; PC1 (or PC3);PC4; PC5 (or PC6A); PC6B; and LPC (or PC7 or PC8). Furin has aubiquitous tissue distribution. It cycles between the trans-Golginetwork (“TGN”), the cell surface, and the endosomes, directed bydefined sequences within furin's cytosolic tail. Furin processes notonly intracellular growth factors and serum proteins, but alsoextracellular matrix proteins and cell surface receptors. Furin has beenreported to cleave proproteins at the consensus sequence-Arg-Xaa-Lys/Arg-Arg-↓ (SEQ ID NO 1). The minimum consensus sequence hasbeen reported to be -Arg-Xaa-Xaa-Arg-↓ (SEQ ID NO 2). See H. Angliker,“Synthesis of tight binding inhibitors and their action on theproprotein-processing enzyme furin,” J. Med. Chem., vol. 38, pp.4014-4018 (1995).

[0005] In addition to these benign physiological roles, furin also playsa role in many pathological pathways, including the cleavage andactivation of bacterial toxins and viral coat proteins, such as toxinsand other proteins from HIV-1 gp160, Newcastle-disease virus_(o),measles virus_(o), human cytomegalovirus glycoprotein B, anthrax toxin,Pseudomonas endotoxin A, diphtheria toxin, and Shiga toxin. Furin hasalso been implicated in assisting the maturation of thematrix-metalloproteinases MT1-MMP and stromelysin-3, a processassociated with metastatic progression in various tumors. Thus non-toxiccompounds that inhibit furin could be useful as a therapeutic agentagainst various bacteria, viruses, and tumors.

[0006] S. Molloy et al., “Bi-cycling the furin pathway: from TGNlocalization to pathogen activation and embryogenesis,” Trends in CellBiology, vol. 9, pp. 28-35 (1999), is a review of the role of furin innumerous biological pathways, including pathogenesis induced by severalbacteria and viruses.

[0007] There have been reports that the P6, P1′ and P2′ positionscontribute to furin catalysis. Like furin substrates, furin inhibitorsalso require that certain subsites be occupied by basic amino acidresidues. For example, the third domain of turkey ovomucoid has beenengineered (KPACTLE¹⁹→KPRCKRE¹⁹) (SEQ ID NOs 3 and 4, respectively) toattempt to increase its specificity towards furin; however the reportedequilibrium constant of 1.1×10⁷ M⁻¹ indicated that it was only amoderate inhibitor. See W. Lu et al., “Arg¹⁵-Lys¹⁷-Arg¹⁸ turkeyovomucoid third domain inhibits human furin,” J. Biol. Chem., vol. 268,pp. 14583-14585 (1993). Inhibition of furin in the sub-nanomolar rangehas been accomplished by bioengineering the reactive site loop of anα1-antitrypsin variant, α1-antitrypsin Portland or α1-PDX, to contain aminimal furin consensus sequence (LEAIMPS³⁵⁹→LERIMRS³⁵⁹) (SEQ ID NOs 5and 6, respectively). Kinetic analysis showed that a portion of boundα1-PDX operates as a tight-binding suicide inhibitor, forming anSDS-stable complex with furin; an alternative pathway involves cleavageand release of α1-PDX. The bait region of the general protease inhibitorα2-macroglobulin (α2M) has been mutated (RVGFYESDVM⁶⁹⁰→RVRSKRSLVM⁶⁹⁰)(SEQ ID NOs 7 and 8, respectively) to attempt to produce a specificfurin inhibitor.

[0008] The ovalbumin-type serpin human proteinase inhibitor8 (PI8),containing two instances of the minimal furin recognition sequence(VVRNSRCSRM³⁴³) (SEQ ID NO 9), has been shown to form SDS-stablecomplexes with furin with an overall K_(i) of 53.8 pM. However, theinhibition of furin by PI8 in vivo, or indeed the co-localization of PI8and furin within the secretory pathway, has not yet been demonstrated.Due to its size, one would expect this proteinase inhibitor to beimmunogenic.

[0009] The only naturally occurring intracellular furin inhibitor thathas been described to date is furin's own propeptide. The prodomains ofproteases often play a role in the activation and regulation of activityof their cognate enzymes. It has been reported that furin is efficientlyinhibited by a GST-furin propeptide fusion construct, and that furinprosegments expressed intracellularly can act in trans to inhibitsubstrate processing.

[0010] The therapeutic value of furin inhibitors was recentlyhighlighted by a report showing that exogenous application of the largeprotein α1-PDX would block in vivo maturation of pro-gB, the humancytomegalovirus envelope glycoprotein. As uptake of α1-PDX into the cellcould not be detected in cell lines lacking the enzyme, it was suggestedthat α1-PDX bound to furin at the cell surface. Pseudomonas exotoxin Aactivation has also been prevented by extracellular application ofα1-PDX to A7 melanoma cells, as has the processing of HIV-1 glycoproteingp160 in transfected cells. These studies demonstrate that the selectiveinhibition of furin can inhibit pathological disease processes.Inhibition of furin can occur on the extracellular surface rather thanin the interior of the cell. It is difficult to obtain α1-PDX in highyield. Due to its size, antitrypsin Portland would be expected to beimmunogenic. See F. Jean et al., Proc. Natl. Acad. Sci. USA, vol. 95,pp. 7293-7298 (1998).

[0011] T. Komiyama et al., “Engineered eglin c variants inhibit yeastand human proprotein processing proteases, Kex2 and furin,” Biochem.,vol. 39, pp. 15156-15165 (2000) reported that certain eglin-basedvariants would inhibit furin. Due to its size, one would expect theeglin protein to be immunogenic.

[0012] Previously reported small molecules that inhibit furin exhibittoxicity at the concentrations needed for inhibition. For example,previously reported inhibitors include decanoyl-RVKR-CH₂-AVG-NH₂ (SEQ IDNO 10) with a Ki of 3.4 nM, ketomethylenes with K_(i)'s in the lowmicromolar range, and the octapeptidyl chloromethane derivativeAc-YEKERSKR-CH₂Cl (SEQ ID NO 11) with a low nM K_(i) for both PC1 andfurin. However, ketones and chloromethane derivatives tend to haveunacceptable in vivo toxicity; hence their use has largely been confinedto probing enzyme-structure relationships in vitro. See, e.g., S.Hallenberger et al., Nature, vol. 360, pp. 358-361 (1992).

[0013] In contrast, polyarginines have been used in vivo for otherpurposes without apparent cytotoxicity, including studies of mucinrelease in goblet cells, activation of phospholipase D, and mimickingthe cationic major basic protein. See K. Ko et al., Am. J. Physiol.,vol. 277, pp. L811-L815 (1999); S. Vepa et al., Am. J. Physiol., vol.272, pp. L608-L613 (1997); A. Coyle et al., Am. J. Respir. Crit. CareMed., vol. 150, pp. S63-S71 (1994); and E. Frigas et al., Mayo Clin.Proc., vol. 56, pp. 345-353 (1981). No prior report has suggested thatpolyarginines should have anti-furin activity.

[0014] L- and D-polyarginines with six or more amino acid residues havebeen reported to enter cells more efficiently than polymers of equallength formed of lysine, ornithine, and histidine. See D. Mitchell etal., “Polyarginine enters cells more efficiently than other polycationichomopolymers,” J. Peptide Res., vol. 56, pp. 318-325 (2000).

[0015] There is an unfilled need for furin inhibitors that combine thecharacteristics of high potency, high stability, high specificity, lowtoxicity, and low molecular weight.

[0016] We have discovered small peptides that strongly inhibit, that arestable, and that have low molecular weight. These peptides are polybasicpeptides, e.g. hexa- to nona-peptides having Arg or Lys in most or allpositions. We also found that removing the peptide terminating groupscan improve inhibition of furin. The most potent inhibitor tested todate, nona-L-arginine (SEQ ID NO 13), had a K_(i) against furin of 42nM. Non-acetylated, poly-D-arginine-derived molecules, e.g.,hexa-D-arginine, are preferred furin inhibitors for therapeutic uses,such as inhibiting certain bacterial infections, viral infections, andcancers. Due to their relatively small size, the peptides used in thisinvention should be non-immunogenic. These peptides are efficientlytransported across cell membranes.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1(a) depicts the results of the purification of recombinantfurin. The figure shows elution volume from the start of the saltgradient.

[0018]FIG. 1(b) depicts a representative chromatogram after thefractions containing the peak enzyme activity were pooled.

[0019]FIG. 2 depicts the effect of pH on furin activity.

[0020]FIG. 3 depicts the inhibition of furin at nanomolar α1-PDXconcentrations.

[0021] FIGS. 4(a) through 4(l) depict the inhibition of furin by variousL-hexapeptides.

[0022] FIGS. 5(a) through 5(f) depict the inhibition of furin by variousD-hexapeptides.

[0023] FIGS. 6(a) and 6(b) depict the K_(i)'s of amidated and acetylatedD- and L-hexapeptides against both furin and PC2.

[0024] FIGS. 7(a) and (b) depict Lineweaver-Burk plots of the mostpotent L- and D-hexapeptides identified from the library screens.

[0025]FIG. 8 depicts the effects of the terminal acetyl and amidemodifications on inhibitory potency.

[0026] FIGS. 9(a)-(d) depict the effect of chain length on theinhibitory properties of L-polyarginine peptides having from 4 to 9arginine residues.

[0027] FIGS. 10(a) through (h) depict the cleavage of nona-L-arginine(SEQ ID NO 13) and hexa-L-arginine (SEQ ID NO 14) by furin.

EXPERIMENTAL PROCEDURES

[0028] Materials. Hexapeptide libraries and synthetic peptides weresynthesized at the Torrey Pines Institute for Molecular Studies (SanDiego, Calif.). Two positional scanning hexapeptide libraries werescreened for inhibition of furin, one made up solely of L-amino acidsand the other solely of D-amino acids. Each hexapeptide librarycomprised 120 peptide mixtures with amino-terminal acetylation andcarboxy-terminal amidation, divided into six groups corresponding toeach position within the hexapeptide. For each position, 20 mixtureswere surveyed, each of which was defined by one of the twenty naturalamino acids. The undefined positions were occupied by any of the aminoacids except cysteine. The positional scanning libraries and theindividual compounds were synthesized using simultaneous multiplepeptide synthesis methods known in the art. The L-polyarginine synthesiswas performed by the Louisiana State University Health Sciences CenterCore Laboratories; mass spectroscopy was used to verify identities ofthe peptides. The α1-PDX was a generous gift from G. Thomas, Portland,Oreg. The anti-furin antiserum, MON148, was a kind gift from W. Van deVen, Leuven, Belgium. Anti-Myc and anti-His antisera were obtained fromInvitrogen (Carlsbad, Calif.). The pERTKR-MCA (SEQ ID NO 12) wasobtained from Peptides International (Louisville, Ky.). N-Glycosidase Fwas obtained from Calbiochem (La Jolla, Calif.).

[0029] Recombinant convertase preparation. The mouse furin clone was akind gift from K. Nakayama (Fukuoka University School of Medicine,Fukuoka, Japan). The mouse furin cDNA was truncated N-terminally to thetransmembrane domain at His⁷¹¹ using PCR. This PCR product was thensubcloned into pcDNA3.1 (−) myc-His (Invitrogen) at the Nhe1 and Xba1restriction sites. Dihydrofolate-reductase-negative DG44 Chinese HamsterOvary (CHO) cells (L. Chasin, Columbia University, New York, N.Y.) weretransfected using Lipofectin (Life Technologies), and colonies wereselected at 37° C. in 5% CO₂ in α-MEM (lacking nucleosides) containing10% well-dialyzed fetal bovine serum (Life Technologies). Conditionedmedia from colonies were screened using an enzyme assay (see below), anda high-expressing clone was selected. Overexpression of furin wasachieved by increasing the methotrexate concentration from 5 nM to 50 μMin five- to ten-fold steps as described in I. Lindberg et al., MethodsNeurosci., vol. 23, pp. 94-108 (1995). The amplified lines were testedfor increased furin expression by enzyme assay. Once the 50 μMmethotrexate level had been reached, cells were split at ratios of 1:6twice a week. 100 mL of conditioned media (OptiMEM, Life Technologies)containing 100 μg/mL aprotinin (Miles Laboratories, Kankakee, Ill.) wascollected from confluent roller bottles every 24 h. The medium was thencentrifuged at low speed to remove cells, and the supernatant was storedat −80° C. until use.

[0030] Purification of furin: Conditioned medium was thawed, pooled, anddiluted 1:3.5 with buffer A (20 mM HEPES, 0.1% Brij 35, 5 mM CaCl₂, pH7.4), and pumped at 40 mL/min through a Sartorius D100 anion exchangemembrane. The membrane was washed with 40 mL of buffer A, followed by 40mL of buffer A containing 50 mM NaCl, and finally by 40 mL of buffer Acontaining 200 mM NaCl. The fraction eluting with 200 mM NaCl wasdiluted 1:4 with buffer A and applied to a 1 mL Pharmacia Mono Q HR5/5anion exchange column at a flow rate of 1 mL/min. Following a 10 mL washwith buffer A, furin was eluted by a linear increase of 0 to 500 mM NaClin buffer A over 30 mL; and 2 mL fractions were collected. Fractionscontaining peak activity were pooled, and 200 μL aliquots were subjectedto gel permeation chromatography using a Pharmacia Superose 12 column ata flow rate of 0.5 mL/min of buffer A containing 200 mM NaCl. Fractionswere assayed for activity as described below; protein content wasdetermined using the Bradford method. All purification steps wereperformed at 4° C.

[0031] Alternatively, the enzyme-containing fraction eluting from theion-exchange membrane with 200 mM NaCl was pumped onto a 1 mL Ni-NTASuperflow (Qiagen) column at 0.3 mL/min, washed with 10 mL of buffer A,and then eluted with a two-step gradient of 0-20 mM imidazole in bufferA over 20 mL, followed by a linear gradient of 20 to 200 mM in imidazolein buffer A. The fractions containing peak enzymatic activity werepooled and subjected to ion exchange chromatography with a Mono Q columnas described above.

[0032] The proprotein convertases PC1 and PC2 were prepared by ionexchange chromatography as described in G. Frenette et al., Biochim.Biophys. Acta., vol. 1334, pp. 109-115 (1997).

[0033] The proprotein convertase PACE4 was partially purified from anovernight-conditioned medium of stably transfected hEK-293 humanembryonic kidney cells (a generous gift of R. E. Mains, Johns HopkinsUniversity School of Medicine, Baltimore, Md.). Briefly, 100 mL ofconditioned medium (OptiMEM containing 100 μg/mL aprotinin) was loadedonto an Econo-Pac Q (Bio-Rad) column at 4 mL/min, washed with 10 mL ofbuffer A, and then eluted with a linear gradient of buffer A containing500 mM NaCl over 50 mL. The active fractions were then diluted 1:4 withbuffer A prior to loading onto a Mono Q column (Pharmacia) at 1 mL/min.The PACE4 was eluted with a linear gradient of buffer A containing 500mM NaCl over 10 mL. The resulting active fractions were then pooled andstored at −80° C. until use. The validity and purity of the preparationwere verified using Coomassie staining and Western blotting with apolyclonal anti-PACE4 antiserum (a gift of R. E. Mains, Johns HopkinsUniversity School of Medicine, Baltimore, Md.).

[0034] Enzyme Assays and Hexapeptide Library Screening. Enzyme assaysfor PC1 and PC2 were performed at pH 5.0 using pERTKR-MCA (SEQ ID NO 12)(Peptides International Inc., Louisville, Ky.) as described in E.Apletalina et al., J. Biol. Chem., vol. 273, pp. 26589-26595 (1998). Theassay for furin was performed using the same substrate at pH 7.0 in 100mM HEPES, 5 mM CaCl₂, 0.1% Brij 35. All assays were performed at 37° C.in a 96 well fluorometer (Labsystems) at an excitation wavelength of 380nm with emission monitored at a wavelength of 460 nm. The total volumewas 50 μL. Unless otherwise stated, the final substrate concentrationfor all assays was 200 μM. When used in a particular experiment, theinhibitory peptides were pre-incubated with enzyme for 30 min at roomtemperature prior to addition of substrate. All assays were performed induplicate or triplicate. Inhibition constants were determined using themethod of Apletalina et al. (1998), and the equationK_(i)=K_(i(app))/(1+([S]/K_(m)). The K_(m)s of PC1, PC2, furin, andPACE4 were determined as 11, 42, 8, and 15 μM, respectively, using acomputerized least squares fitting technique with EnzFitter (BioSoft,Cambridge, England).

[0035] Digestion of recombinant furin with N-Glycosidase F. A 200 μLaliquot of the pooled fractions from the Superose chromatography(containing 40 μg of furin) was made up to 4.5% beta mercaptoethanol,0.45% SDS and boiled for ten minutes prior to concentration to 60 μLusing a Centricon 10 (Amicon). The concentrate was diluted to 400 μLusing 50 mM sodium phosphate buffer, pH 7.5, 0.76% Triton X-100; and 1.8μg of N-Glycosidase F (Calbiochem) was added. The sample was incubatedat 37° C., and 45 μL aliquots were removed at the times indicated andplaced in 5 μL of 5×SDS buffer prior to boiling for 3 min. The aliquotswere separated by SDS-PAGE (8.8%) and visualized with Coomassie bluestaining.

[0036] Cleavage of Nona-L-arginine and Hexa-L-arginine by Furin.Nona-L-arginine (200 μM) (SEQ ID NO 13) or hexa-L-arginine (200 μM) (SEQID NO 14) was incubated at 37° C. with or without furin (1.7 μM), in 100mM HEPES, pH 7 containing 5 mM CaCl₂ and 0.2% Brij 35. Aliquots (20 μL)were removed at the indicated times, placed into 480 μL ice-cold 0.1%TFA, immediately frozen and kept frozen until HPLC analysis. Afterthawing, the aliquots were separated on a 5 μm, 0.46×25 cm Beckman(Fullerton, Calif.) ODS column with a linear gradient of 0 to 15%acetonitrile containing 0.1% trifluoroacetic acid over 40 min at 1mL/min. Absorbance was monitored at 214 nm. Cleavage products wereidentified by comparison to polyarginine standards. Parallel reactionscontaining buffer instead of furin were also analyzed.

[0037] Results

[0038] Overexpression, Purification, and Characterization of RecombinantMouse Furin. The use of the dihydrofolate reductase-coupledamplification method to overexpress truncated furin produced a cell linethat secreted roughly 0.8 μg/mL furin into the culture medium, asestimated by the specific activity of the purified protein. As shown inTable 1, the initial ion exchange step, while having a relatively lowyield, nevertheless proved valuable as a method of rapidly concentratingthe conditioned medium from a large volume, while at the same timeremoving phenol red and contaminating protein from the product. Afterthe volume was thus reduced, it was then possible to load the highresolution Mono Q (Pharmacia) ion exchange column used in the second ionexchange step within a reasonable time.

[0039]FIG. 1(a) depicts the results of the purification of therecombinant furin. Partially purified, concentrated recombinant mousefurin from the first ion exchange step was diluted 1:4 with buffer A andpumped through a MONO Q HR5/5 column (Pharmacia) equilibrated withbuffer A. The column was washed with 5 mL of buffer A before elutionwith a 30 mL gradient of 0 to 500 mM NaCl in buffer A. The figure showselution volume from the start of the salt gradient.

[0040]FIG. 1(b) depicts a representative chromatogram after thefractions containing the peak enzyme activity were pooled and thealiquots applied to a Superose 12 column. The bars depict activityagainst pERTKR-MCA (SEQ ID NO 12). The solid line depicts UV absorbanceat 280 nm. The gel permeation column was calibrated with the molecularweight standards marked as □: thyroglobulin, 670 kDa; IgG, 150 kDa;ovalbumin, 44 kDa; myoglobin, 17 kDa; cyanocobalamin, 1.35 kDa. (Allmolecular weight standards were obtained from Biorad).

[0041] As can be seen in FIG. 1(a), the majority of the protein elutedas a single peak, coincident with the proprotein convertase activity.Again, the yield from this step was low, but an appreciable increase inspecific activity was observed. See Table 1. TABLE I Purification ofRecombinant Furin Total Total Specific Purification Activity ProteinActivity Yield Purification Step (Units^(a)) (mg) (units/mg) (%) FactorConditioned 156  54 2.9 (100) (1) Medium Ion 84 7.6 11 54 3.8 Exchange 1Ion 46 2.4 19 29 6.7 Exchange 2 Gel 42 2 21 27 7.2 Permeation

[0042] The fractions with maximum activity were pooled and subjected togel permeation chromatography, as shown in FIG. 1(b). In this stepvirtually all the protein eluted as a single peak, with only smallamounts eluting at lower and higher molecular weights. The furinactivity exactly coincided with the major absorbance peak. The fractionshaving maximum activity were pooled, diluted with glycerol to a finalconcentration of 10%, and stored at −80° C. until use. Under theseconditions there was no detectable loss in activity over six months.Molecular weight standards used to calibrate the gel permeation columnindicated a molecular weight for furin of about 59 kDa. Coomassie Bluestaining and Western blotting (data not shown) of the gelpermeation-purified fractions revealed a single band at 61 kDa. Thefinal specific activity was 21 Units/mg protein; and the overall yieldfor the purification was 27%, with a purification factor of 7.2.

[0043] We had initially attempted purifications using a C-terminallylocated hexa-His (SEQ ID NO 15) tag as a ligand for affinitychromatography with a metal ion chelation resin (Ni-NTA Superose,Qiagen). However, the furin activity then eluted at very low (˜20 mM)imidazole concentrations, with no increase in specific activity comparedwith the sample applied. Subsequent Western blotting with both anti-Hisand anti-Myc antisera (Invitrogen) showed no immunoreactivity, whereasblotting using the anti-furin antiserum Mon148 (a generous gift of W.Van de Ven, University of Leuven, Leuven, Belgium) revealed a strongband at 61 kDa (not shown), indicating that C-terminal truncation of thesecreted product had occurred. The metal ion chelation step wassubsequently abandoned and all other data presented here were obtainedusing furin purified using ion exchange and gel permeationchromatography as otherwise described above.

[0044] Treatment of the purified furin with N-glycosidase F revealed thepresence of two lower molecular weight forms, indicating that two of thethree potential sites in the recombinant furin preparation were presentand originally glycosylated.

[0045]FIG. 2 depicts the effect of pH on furin activity. While there wasa rapid drop in activity below pH 6.5, the enzyme retained greater than90% of maximum activity at pH 9.0. Calcium concentrations over 1 mM wererequired for full activity, with no significant difference in activityas calcium concentrations were increased thereafter to 50 mM. Consistentwith previous reports, as shown in FIG. 3, furin was strongly inhibitedat nanomolar α1-PDX concentrations, giving further validation of theenzyme preparation.

[0046] L-Hexapeptide Library Scan. To identify amino residues playing asignificant role in the inhibition of furin, we screened a positionalscanning L-hexapeptide library (amino terminally acetylated and carboxyterminally amidated) using the standard enzyme substrate pERTKR-MCA(i.e., pyr-Glu-Arg-Thr-Lys-Arg-methylcoumarinamide) (SEQ ID NO 12). Intotal, the library was screened nine times, at inhibitor concentrationsof 1.0 and 0.5 mg/mL, and at substrate concentrations of 200 and 100 μM.The concentrations of inhibitor and substrate were found to influencethe degree of observed inhibition. Screening at the lower substrateconcentration gave better discrimination between peptides bearingdifferent residues at all inhibitor concentrations. In addition, at thelower substrate concentration, better discrimination was shown forpositions P1, P2 and P3 at 1 mg/mL inhibitor concentration (FIGS.4(a)-(f)), while better discrimination for positions P4, P5 and P6 wasseen at 0.5 mg/mL inhibitor concentration (FIGS. 4(g)-(l)).

[0047] FIGS. 4(a) through 4(l) depict the inhibition of furin by variousL-hexapeptides. Each peptide mixture was pre-incubated with furin inassay buffer for 30 min prior to the addition of substrate (finalconcentration, 100 μM). The rate of hydrolysis of pERTKR-MCA wasfollowed for 1 hour. Inhibition is given as the percentage decrease inactivity in the presence of the peptide mixture relative to that ofcontrol. In FIGS. 4(a) through 4(f), the peptide concentration was 1mg/mL. In FIGS. 4(g) through 4(l), the peptide concentration was 0.5mg/mL.

[0048] From FIGS. 4(a)-(f) (i.e., the experiments using an inhibitorconcentration of 1 mg/mL) it can be seen that at position P1, Arg, Lysand His exerted greater than average inhibition, while at positions P2and P3, Arg and Lys, but not His, were the preferred residues. Atpositions P4, P5 and especially at position P6, many residues showedgreater than average inhibition, but no clear distinction could be madeon either the basis of size, hydrophobicity or charge. In contrast, atthe lower 0.5 mg/mL inhibitor concentration (FIGS. 4(g)-(l)), whilethere were no clearly preferred residues at positions P1, P2 or P3,there was good discrimination at positions P4, P5 and P6. On the basisof the screens shown in FIG. 4, we selected Arg in positions P1, 2 and3, Lys in P4; His or Arg in P5; and His, Met, Lys or Arg in P6 forassembly into discrete peptide sequences.

[0049] D-Hexapeptide Library Scan. The positional scanning acetylatedand amidated D-hexapeptide library was screened a total of nine times ateither 0.5 mg/mL or 1.0 mg/mL inhibitor and either 50 μM or 100 μMsubstrate concentration; in all cases the results were similar. Arepresentative screen is shown in FIGS. 5(a)-(f).

[0050] FIGS. 5(a) through 5(f) depict the inhibition of furin by variousD-hexapeptides. Each peptide mixture (final concentration, 1 mg/mL) waspre-incubated with furin in assay buffer for 30 min prior to theaddition of substrate (final concentration, 100 μM). The rate ofhydrolysis of pERTKR-MCA was followed for 1 hour. Inhibition is given asthe percentage decrease in activity in the presence of the peptidemixture relative to that of control.

[0051] While D-Arg was one of the preferred residues in all positions,the remainder of the inhibitory residues were hydrophobic.Interestingly, D-Lys effected greater than average inhibition only inposition P6, but still showed less inhibition than the most effectiveresidue, D-Trp. In positions P3, P4, and P5, D-Arg was marginally themost potent residue when all results were averaged. In position P2,D-Arg and D-Ile consistently produced relatively high inhibition, whilethe same was true for D-Arg and D-Leu in position P1. In all casesrelative inhibition values were consistently lower than those of theL-hexapeptide library, indicating a preference of furin for L-residues.Nevertheless, a series of D-hexapeptides was synthesized based upon theresults described above, in which position P1 was either D-Arg or D-Leu,P2 was either D-Arg or D-Ile, positions P3, P4 and P5 were always D-Arg,and P6 was always D-Trp.

[0052] Inhibition of Furin and PC2 by Synthetic Peptides. The amidatedand acetylated D- and L-hexapeptides that were synthesized based on theresults of the D- and L-hexapeptide library screens were tested againstboth furin and PC2. For both the D- and L-peptide series, the K_(i)'sagainst furin were all in the low μM range; these peptides all inhibitedfurin ˜10-100 times more strongly than they inhibited PC2. (See Table 2and FIGS. 6(a) and 6(b)). Against furin, the potency increased as thesequence became more basic, an observation that did not hold for PC2.Examination of the K_(i)'s of the L-hexapeptides against furin revealedthat in position 5, Arg was preferred to His, and that the inhibitorypotency of these peptides against furin increased as P6 was changed inthe order His, Met, Lys, Arg. The same order of inhibitory potency wasnot seen against PC2: in this instance His was preferred to Arg in P5.While the combination of a P5 Arg and a P6 Met was severely unfavorableas a PC2 inhibitor, when used against furin the influence of the P5residue appeared to outweigh that of the P6 residue.

[0053] The D-hexapeptide inhibitors were also assayed against furin andPC2 (Table 2). Against furin, D-Arg was preferred to D-Ile in P2, andD-Arg was preferred to D-Leu in P1. However, the presence of a basicresidue at P1 or P2 was sufficient to produce a relatively potent furininhibitor, despite the presence of a hydrophobic residue at P2 or P1.Conversely, when D-Arg was present at P2, substituting D-Leu for D-Argin P1 produced a more potent PC2 inhibitor. Thus, like the L-peptideinhibitors, increasing basicity resulted in a more potent furininhibitor, but not a more potent PC2 inhibitor. TABLE 2 Inhibitionconstants of various L- and D- hexapeptides against furin and PC2^(b)K_(i) (μM) Furin PC2 L-Peptides Ac-HHKRRR-NH₂ 13.2 ± 1.6  235 ± 16 (SEQID NO 16) Ac-MHKRRR-NH₂ 10.3 ± 1.4  216 ± 13 (SEQ ID NO 17)Ac-KHKRRR-NH₂ 5.2 ± 0.9 280 ± 29 (SEQ ID NO 18) Ac-RHKRRR-NH₂ 3.4 ± 0.6152 ± 30 (SEQ ID NO 19) Ac-HRKRRR-NH₂ 2.1 ± 0.5 309 ± 29 (SEQ ID NO 20)Ac-MRKRRR-NH₂ 2.3 ± 0.5 1,500 ± 300  (SEQ ID NO 21) Ac-KRKRRR-NH₂ 1.6 ±0.5 391 ± 60 (SEQ ID NO 22) Ac-RRKRRR-NH₂ 1.3 ± 0.9 461 ± 75 (SEQ ID NO23) D-Peptides Ac-wrrril-NH₂ 22.7 ± 4.3   601 ± 200 Ac-wrrrir-NH₂ 7.0 ±0.9 399 ± 75 Ac-wrrrrl-NH₂ 5.3 ± 1.0 203 ± 20 Ac-wrrrrr-NH₂ 2.4 ± 0.8334 ± 37

[0054] Mechanism of Inhibition. FIGS. 7(a) and (b) depictLineweaver-Burk plots of the most potent L- and D-hexapeptidesidentified from the library screens. Furin (30 nM) was pre-incubated in100 mM HEPES, 5 mM CaCl₂, 0.1% Brij 35, pH 7.0 with either 0 (▪), 20 ()or 40 (▴) μM of Ac-RRKRRR-NH₂ (FIG. 7(a)) (SEQ ID NO 23) or ofAc-wrrrrr-NH₂ (FIG. 7(b)) prior to addition of substrate at the finalconcentrations indicated. The results demonstrated strictlycompetitive-type inhibition for both the L- and D-peptides. No deviationwas seen from classical Michaelis-Menten-type kinetics, typical of tightbinding or suicide inhibitors such as displayed by α1-PDX and thechloromethyl derivatives, both of which function by forming anirreversible complex with the enzyme.

[0055] The Effect of N-Terminal Acetyl and C-Terminal Amide Groups onInhibition. We also examined the effect of the terminal acetyl and amidemodifications on inhibitory potency. Initially, various forms of anamidated and acetylated L-hexapeptide, LLRVKR (SEQ ID NO 24), previouslyidentified by Apletalina et al. (1998) as a nanomolar inhibitor of PC1,were tested against furin and PC2. The results are shown in Table 3 andFIG. 8. Interestingly, removing the terminal amide and acetyl groupsfrom this hexapeptide increased its inhibitory potency against furineight-fold. It appeared that the relative lack of inhibitory potency ofthe unacetylated and unamidated peptide against furin was almost solelyattributable to the N-terminal acetyl group. In contrast, when the samepeptides were tested against PC2, the terminating groups appeared toassist in inhibition. The K_(i) of the acetylated and amidated peptidewas nearly four-fold smaller against PC2 than the K_(i) of theunmodified peptide. Comparing peptides, it was seen that removing theN-terminal acetyl group resulted in the loss of inhibitory potencyagainst PC2, implying that PC2 has sequence recognition ability thatextends beyond the P6 side chain. TABLE 3 The effect of N-terminalacetylation and C- terminal amidation on the inhibition of furin andPC2^(c) by SEQ ID NO 24 K_(i) (μM) Furin PC2 LLRVKR-NH₂ 0.8 ± 0.1 2.3 ±0.2  Ac-LLRVKR 3.5 ± 0.2 1.3 ± 0.6  Ac-LLRVKR-NH₂ 3.4 ± 0.1 1.0 ± 0.08LLRVKR 0.42 ± 0.02 3.7 ± 0.17

[0056] Inhibition of Furin, PACE4, PC1 and PC2 by DifferentL-Polyarginine Peptides. A series of L-polyarginine peptides, with chainlengths of 4 to 9 residues and no terminal modifications, wassynthesized and tested for inhibitory potency against furin, PC1, PC2and PACE4. Table 4 and FIGS. 9(a)-(d) show that the K_(i) of theL-polyarginine peptides against furin increased from 42 nM to 6 μM asthe chain length decreased from 9 to 4 residues. While the K_(i)'s ofthe nona-, octa-, hepta-, and hexamers ranged from 42 to 114 nM, therewas an approximate 10-fold increase in K_(i) between the hexa- andpentamer, and about a five-fold increase between the penta- andtetramer. TABLE 4 Inhibition constants of various polyarginine peptidesagainst furin, PACE4 and PC1^(d) K_(i) (μM) Furin PACE4 PC1Tetra-L-arginine 6.4 ± 0.9 >200 >200 (SEQ ID NO 25) Penta-L-arginine0.99 ± 0.08 0.98 ± 0.120  14 ± 6.1 (SEQ ID NO 26) Hexa-L-arginine 0.114± 0.006 0.52 ± 0.045  3.9 ± 0.62 (SEQ ID NO 14) Hepta-L-arginine 0.068 ±0.001 0.24 ± 0.045 5.2 ± 1.2 (SEQ ID NO 27) Octa-L-arginine 0.061 ±0.001 0.15 ± 0.060 5.1 ± 2.0 (SEQ ID NO 28) Nona-L-arginine 0.042 ±0.003 0.11 ± 0.013  12 ± 2.5 (SEQ ID NO 13) Hexa-D-arginine 0.106 ±0.010 0.58 ± 0.040   13 ± 0.25

[0057] The K_(i)'s against PACE4 also increased as the chain length ofthe inhibitor decreased, but unlike furin, the K_(i) of the pentamericpolyarginine (SEQ ID NO 26) was approximately twice that of the hexamer(SEQ ID NO 14), and no sharp change was seen as the chain length wasreduced below the n=6 level. The minimum K_(i) observed was 110 nM. Thetetramer (SEQ ID NO 25), however, was not found to inhibit even at mMconcentrations. By contrast, the polyarginine peptides were onlymoderate inhibitors of PC1, with a minimum K_(i) of ˜4 μM.Interestingly, the K_(i) of the nonamer (SEQ ID NO 13) was significantlygreater than those of the hexa-, hepta-, and octamer (SEQ ID NOs 14, 27,and 28, respectively). Similarly to PACE 4 (but not to furin), PC1 wasnot inhibited by tetra-L-arginine (SEQ ID NO 25) at μM concentrations.Overall, it appeared that the binding pockets of furin and PACE4 weremore similar to each other than either was to that of PC1, but thatfurin has a unique dependence on the S6 binding pocket. (The “S6 bindingpocket,” in standard nomenclature, is that part of the enzyme that bindsthe sixth residue of the substrate, counting backward from the scissilebond.)

[0058] By contrast to the other proprotein convertases studied here, PC2activity was consistently stimulated by the polyarginine peptides. Thestimulatory effect was noticeable with all polyarginines tested,starting at concentrations as low as 0.1 nM and increasing withconcentration up to approximately 10 μM of peptide, following which arelative decrease in activity was observed (data not shown). No effectcould be confidently correlated with the peptide length for PC2, exceptat low nM peptide inhibitor concentrations, where the tetra- andpenta-L-arginines (SEQ ID NOs 25 and 26) appeared to produce a smallerstimulatory effect did than the longer peptides (data not shown).

[0059] In addition, hexa-D-arginine was synthesized and tested forinhibitory potency against furin, PACE4, PC1, and PC2. The K_(i)'sagainst furin and PACE4, as shown in Table 4, were remarkably similar tothose for hexa-L-arginine, while against PC1 a three-fold increase inK_(i) was observed. When tested against PC2, no stimulatory orinhibitory effect was observed (results not shown).

[0060] Mechanism of Inhibition. Lineweaver-Burk plots forhexa-L-arginine (SEQ ID NO 14), nona-L-arginine (SEQ ID NO 13), andhexa-D-arginine (not shown) demonstrated that, like the acetylated andamidated hexapeptides shown in FIG. 4, these compounds demonstratedstrictly competitive-type inhibition. The concentrations of polyarginineused to generate these data were forty-fold lower than theconcentrations of amidated and acetylated hexapeptides used in FIG. 4.

[0061] Cleavage of Nona-L-arginine (SEQ ID NO 13) and Hexa-L-arginine(SEQ ID NO 14) by Furin. FIGS. 10(a) through (h) depict the cleavage ofnona-L-arginine (SEQ ID NO 13) and hexa-L-arginine (SEQ ID NO 14) byfurin. Furin (FIGS. (10(b), (c), (d), (e), and (g)) or buffer (FIGS.10(f) and (h) was incubated with nona-L-arginine (SEQ ID NO 13) (FIGS.10(b), (c), (d), (e), and (f) or hexa-L-arginine (SEQ ID NO 14) (FIGS.(10(g) and (h) at 37° C. for 0 min (FIG. (10(b)), 40 min (FIG. 10(c)), 6h (FIG. 10(d)), or 24 h (FIGS. 10(e), (f), (g), and (h) prior toseparation by HPLC as described in the experimental procedures. In FIG.10(a) the simple separation of a standard mixture polyarginines (withoutenzyme) is shown; the number of residues per poly-L-arginine isindicated by the positions of the arrows at the tops of FIGS. 10(a) and(e).

[0062] Cleavage of nona-L-arginine (SEQ ID NO 13) was first observed ˜40min after reaction with furin had commenced, with the appearance ofhexa- and hepta-L-arginine (FIG. 10(c)) (SEQ ID NOs 14 and 27). Afterfour hours penta-L-arginine (SEQ ID NO 26) was also observed, andessentially none of the original nona-L-arginine (SEQ ID NO 13)remained. The heptapeptide (SEQ ID NO 27) was still present after sixhours of digestion (FIG. 10(d)), but after 24 hours, essentially onlythe penta- and hexapeptides (SEQ ID NOs 26 and 14) were present (FIG.10(e)). No significant amount of tetra-L-arginine (SEQ ID NO 25) wasobserved at any time. Cleavage of hexa-L-arginine (SEQ ID NO 14)proceeded much less rapidly than did that of nona-L-arginine (SEQ ID NO13); indeed essentially no cleavage was seen after six hours incubationwith furin (results not shown). After 24 hours, partial digestion ofhexa-L-arginine (SEQ ID NO 14) had occurred, producing somepenta-L-arginine (SEQ ID NO 26) (FIG. 10(g)). Again, essentially notetra-L-arginine (SEQ ID NO 25) product was seen. Controls, where bufferreplaced furin, are shown in FIGS. 10(f) and 10(h), each after a 24 hincubation at 37° C.

[0063] These data show that L-polyarginine is preferentially orientedinto the catalytic pocket of furin such that side chains interact withthe S1-S6 binding pockets. When the experiment was repeated withhexa-D-arginine and furin, or with nona-L-arginine (SEQ ID NO 13) andPC2, essentially no cleavage was observed after 24 h of incubation(results not shown).

[0064] Discussion

[0065] We have purified and partially characterized a recombinant,truncated mouse furin from the conditioned medium of CHO cells. Ourpurified furin preparation was homogeneous, with an apparent molecularweight of 61 kDa by SDS-PAGE and 59 kDa by gel permeationchromatography. The enzyme was shown to be C-terminally processed, asthe C-terminally-located tags could not be detected by Western blotting,giving a molecular weight of approximately 60 kDa. Treatment withN-glycosidase F suggested that this furin was glycosylated at two ofthree potential sites. A truncated furin preparation had previously beenshown to be C-terminally processed, with a similar 5 kDa shift inapparent mobility on SDS-PAGE following N-glycosidase F; however, to thebest of our knowledge, this is the first time the number ofglycosylation sites has been demonstrated. The specific activity of thepurified enzyme against pERTKR-MCA (SEQ ID NO 12) of 21 μmol AMC/h wassimilar to that in a previous report of a maximum specific activity of30 μmol AMC/h using Boc-RVRR-MCA. The overall yield of 27% wasrelatively low, probably reflecting the use of two ion-exchange steps inour protocol.

[0066] The enzyme suffered only a slight loss of activity at pH 9.0. ThepH dependence of furin may depend on the source, substrate, and degreeof purification. The purified enzyme used in this study was stronglyinhibited by the furin-specific serpin α1-antitrypsin-PDX atconcentrations identical with those previously reported.

[0067] Basic Residues in All Positions Favor Inhibition of Furin, butnot of PC1 and PC2

[0068] By contrast to results our laboratory had previously obtained forL-hexapeptide combinatorial library screens against PC1 and PC2, furinrevealed a preference for Arg and Lys in all six positions, with Argbeing the more inhibitory of the two residues in all positions exceptP4. In addition, some preference was also shown for His in positions P1,P4, P5 and P6. In contrast, our previous work with PC1 had shown astrong preference for Arg in P1 and P4, Lys in P2, and Leu in P6, whilethe P3 and P5 residues could be interchanged with relatively littleeffect on inhibition. Screens against PC2 showed that Arg in positionsP1 and P4 consistently gave the highest inhibition, while at the otherfour positions no clear consensus was seen. Thus it appears that thebinding pocket of furin, unlike that of PC1 and PC2, has a preferencefor basic residues that stretches from the S1 to the S6 subsites. M.Zhong et al., J. Biol. Chem., vol. 274, pp. 33913-33920 (1999) showedthat peptides based on the prodomain sequences of both furin and PC7could act as potent inhibitors of either enzyme: The furin propeptidecould be reduced to a ten-residue sequence (QQVAKRRTKR) (SEQ ID NO 29),with a K_(i) of 40 nM against furin and approximately 500 nM againstPC7. When the C-terminal residue was changed to a non-basic alanine,inhibitory potency was abolished. A decapeptide fragment (EQRLLKRAKR)(SEQ ID NO 30) of the propeptide of PC7 showed a K_(i) of 80 nM towardsfurin and 6 nM against PC7. It should be noted, however, thatdifferences in the furin preparation and in the methods used tocalculate the K_(i)'s preclude direct comparison of our numerical valueswith those of Zhong et al. Nonetheless, our results show that theinhibitory potency of peptides against furin is correlated with theconcentration of positive charges, and indicate that this may be aselective property of furin.

[0069] D-Residues can be Used to Construct Relatively Potent InhibitoryPeptides

[0070] Although the D-hexapeptide screen showed somewhat lowerinhibition of furin than did the L-hexapeptide library, the K_(i)'s ofthe synthetic D-peptides were surprisingly similar to those of theL-peptides (Table 2), indicating a similar mechanism of inhibition. AsD-peptides should be more resistant to hydrolysis than L-peptides invivo, the D-peptides may have greater stability for use as a therapeuticfurin inhibitor. D-peptides are completely resistant to hydrolysis byfurin.

[0071] Furin and PC2 are Sensitive to Groups Distal to the P1 and P6Residues

[0072] We have shown above that furin is sensitive to groups locatedtowards the C-terminal from the P1 side-chain, with a doubling of theK_(i) upon C-terminal amidation of hexapeptides. We have also shown thatfurin is sensitive to groups distal to the P6 side chain; N-terminalacetylation of an L-hexapeptide increased its K_(i) by a factor ofeight. These results are consistent with the data of D. Krysan et al.,J. Biol. Chem., vol. 274, pp. 23229-23234 (1999), who showed substrateinhibition with hexa- but not tetrapeptide substrates. In the same studya comparison of furin with the related proprotein convertase Kex2revealed that while the residue at the P1 position had a large effect oncatalysis, the P4 and P6 residues were especially important for furin.Furthermore, favorable residues at P2 and P6 were able to compensate forless than optimal residues at P1 and P4. Our data indicated that theeffect of acetylation and amidation on the inhibition of PC2 was theopposite of that for furin. However, like furin, P6 acetylation of PC2inhibitors had the largest single effect on inhibition, demonstratingthat, like furin, the binding pocket of PC2 extends beyond the P6residue.

[0073] Hexa-L-Arginine (SEQ ID NO 14) is a Potent Inhibitor of Furin,but Stimulates PC2 Activity.

[0074] In U. Shinde et al., Semin. Cell Dev. Biol., vol. 11, pp. 35-44(2000); and A. Basak et al., Int. J. Pept. Protein Res., vol. 44, pp.253-261 (1994), peptides corresponding to known substrate cleavage siteswere used as starting points for the synthesis of peptide inhibitors offurin. A series of deca- and dodecapeptides based upon a partialsequence of the junction between the propeptide domain and the catalyticdomain of PC1 were tested for inhibition of PC1 and furin. Thesepeptides contained a variety of unnatural amino acids in the P′1position. Interestingly, the compounds were found to be slightly betterinhibitors of furin than of PC1, with K_(i)'s for the dodecapeptidesranging from 0.8 to 10 μM for furin, compared to 1.0 to 170 μM for PC1.The K_(i)'s of the decapeptides ranged from 1.0 to 8.6 μM against PC1,and from 0.8 to 2.2 μM against furin. While the K_(i) of the ten-residuepropeptide fragment identified by Zhong et al. (1999) was essentiallythe same as the K_(i) of nona-L-arginine (SEQ ID NO 13), if cleavage atthe P3-P2 bond were to occur, as with nona-L-arginine, the resultingfragment, QQVAKRRT (SEQ ID NO 31), would be expected to have littleinhibitory ability due to the lack of a basic residue at P1. Incontrast, we observed that cleavage of nona-L-arginine (SEQ ID NO 13)results in peptides having K_(i)'s in the low nanomolar range.

[0075] A comparison of inhibition of the proprotein convertases PC1,PC2, furin and PACE4 with polyarginine derivatives revealed strikingdifferences. Whereas furin and PACE4 were both inhibited toapproximately the same extent by all polyarginines tested excepttetra-L-arginine (SEQ ID NO 25), PC1 was much less sensitive to thepeptides than was furin, while PC2 was consistently stimulated. Theseresults suggest that the binding pocket of PACE4 is relatively similarto that of furin.

[0076] It has been previously observed that PC2 is fundamentallydifferent from the other members of the proprotein convertase family,for example being the only member requiring the presence of theneuroendocrine protein 7B2 for full activity; activating late in thesecretory pathway; and possessing an Asp rather than an Asn in theoxyanion hole. The stimulation of PC2 by L-polyarginines that weobserved was not due to more rapid activation of the recombinant proPC2,as maximum activity was attained within 30 min of a reduction in pH from7.4 to 5.0, irrespective of the presence of polyarginine, and theactivity then remained constant for 90 min thereafter (results notshown). Thus it appeared that either a greater proportion of the enzymepreparation was activated by interactions at an allosteric site, or thepolyarginine peptides somehow directly assist substrate turnover.

[0077] The Furin Catalytic Pocket: Differences with PC1 and PC2

[0078] As the polyarginines tested contained the furin cleavageconsensus sequence, we expected cleavage to occur in at least some ofthe polyarginines, an expectation that was confirmed by experimentalobservations. However, while nona-L-arginine (SEQ ID NO 13) was indeedcleaved by furin, the two primary products were the hexamer (SEQ ID NO14) and the heptamer (SEQ ID NO 27); the penta-L-arginine (SEQ ID NO 26)product observed after 4 hours (FIG. 10(e)) was most likely due tofurther cleavage of the heptamer (SEQ ID NO 27), as incubation of thehexamer (SEQ ID NO 14) with furin only produced the pentamer (SEQ ID NO26) after 24 hours. These results demonstrated that furin does notcleave hexa-L-arginine (SEQ ID NO 14) at the P2 position at asignificant rate, an important finding for inhibitors to be used invivo, in vitro, or ex vivo. It is also interesting that furin showed anabsolute preference for substrates having five or six residuesN-terminal to the cleavage site over substrates having only two, three,or four residues N-terminal to the cleavage site. This implies that theS6 binding pocket of furin is as important to specificity as are thesites closer to the catalytic triad.

[0079] Taken together, our results imply that the furin subsites allappear to be negatively charged, as opposed to those of PC1 and PC2,whose S3 and S6 subsites apparently use hydrophobic interactions, stericinteractions, or both. The similar specificity of the polyargininesagainst PACE4 and furin agrees with observations that these twoproprotein convertases are more closely related to each other, bothstructurally and spatially, than to either PC1 or PC2.

[0080] Polyarginines as Therapeutically Useful Furin Inhibitors

[0081] Polyarginines, both L- and D-forms, are potent and relativelyspecific furin inhibitors. We do not expect therapeutic uses of thesepeptides to be substantially affected by the ability of such highlycharged molecules to cross the cell membrane unaided, because one of thedefining features of furin is its ability to cycle between the TGN, thecell surface, and the endosomes. For example, it has been shown thatα1-PDX can be internalized by cells producing furin, but not byfurin-deficient cells. We expect that the internalization ofpolyarginines by cell-surface exposed furin to be efficient, given theirsmall size and solubility compared to α1-PDX.

[0082] Compared to other low molecular weight proprotein convertaseinhibitors that have been reported, our preliminary data show thatpolyarginines have low toxicity at the concentrations needed to inhibitfurin. In particular, our results show that the D-polyargininehexapeptide (D6R) was not toxic to cells, and that it is able to protectcells from killing by diphtheria exotoxin A. These preliminary data arediscussed briefly below, and are summarized in Tables 5 and 6.

[0083] HEK293 cells were seeded into 96-well plates at the densitiesindicated. Their growth rates were monitored using the dye WST, which iscleaved by mitochondrial dehydrogenases to a blue dye only in viablecells. The mean absorbance of three wells per condition at 450 nm, plusor minus the standard deviation, is given in Table 5. Table 5 shows thatthe addition of D-hexa-arginine (D6R) at the concentrations shown didnot significantly affect the growth of the cells. We concluded that D6Rdid not appear to be cytotoxic even at 100 μM (final concentration).TABLE 5 Life curve Time 5 × 10² 2 μM 4 μM 6 μM 8 μM 10 μM 30 μM 60 μM100 μM (hour) cells/well D6R D6R D6R D6R D6R D6R D6R D6R 2.5 0.05 ± 0.0124 0.27 ± 0.04 48 0.63 ± 0.02 51 0.65 ± 0.03 0.69 ± 0.64 ± 0.63 ± 0.67 ±0.64 ± 0.64 ± 0.66 ± 0.62 ± 0.02 0.03 0.04 0.01 0.03 0.02 0.05 0.03 751.25 ± 0.02 1.25 ± 1.28 ± 1.13 ± 1.36 ± 1.28 ± 1.29 ± 1.17 ± 1.19 ± 0.110.14 0.14 0.08 0.14 0.08 0.08 0.11 99 1.46 ± 0.05 1.42 ± 1.39 ± 1.48 ±1.40 ± 1.49 ± 1.45 ± 1.48 ± 1.47 ± 0.02 0.06 0.04 0.05 0.08 0.06 0.060.09

[0084] Pseudomonas exotoxin A (PEA) must be cleaved by furin at the cellsurface to gain entry into a cell, which typically causes cell death.Table 6 shows that the addition of PEA to logarithmically growing HEK293cells at a concentration of 10 ng/mL caused death of many cells,evidenced by a decrease in the amount of WST at all times, as comparedto wells lacking PEA. However, adding D-hexa-arginine at 1 μM finalconcentration reduced the cell death caused by PEA. We inferred that theD6R blocked the cleavage of PEA by furin, thus preventing itsactivation. TABLE 6 100 μM Life curve 0 D6R 1 μM D6R 10 μM D6R D6R Time5 × 10² cells/ 0 D6R 10 ng/mL 10 ng/mL 10 ng/mL 10 ng/mL (hours) well 0PEA PEA PEA PEA PEA 2.5 0.09 ± 0.01 24 0.26 ± 0.03 48 0.45 ± 0.02 510.53 ± 0.02 0.58 ± 0.02 0.46 ± 0.03 0.52 ± 0.04 0.54 ± 0.07 0.51 ± 0.0476 1.00 ± 0.06 1.03 ± 0.09 0.63 ± 0.01 0.85 ± 0.02 0.73 ± 0.05 0.78 ±0.03 86 1.15 ± 0.01 1.17 ± 0.04 0.38 ± 0.04 0.83 ± 0.02 0.71 ± 0.02 0.61± 0.06 96 1.65 ± 0.05 1.65 ± 0.03 0.21 ± 0.02 0.56 ± 0.04 0.67 ± 0.010.55 ± 0.02

[0085] Therapeutic Applications

[0086] The administration of polyarginines and other polybasic peptidesin accordance with the present invention may be used to combat bacterialand viral infections, and to inhibit the growth of certain cancers.Preliminary data, for example, show activity against Pseudomonasexotoxin, and against HIV. For example, preliminary data (not shown)suggested that micromolar concentrations of D-hexa-arginine inhibitedthe formation of syncytia by HIV in vitro in the MT4 line of T cells.

[0087] Furin is thought to play a role in the pathogenesis of manyviruses and bacteria. See S. Molloy et al., “Bi-cycling the furinpathway: from TGN localization to pathogen activation andembryogenesis,” Trends in Cell Biology, vol. 9, pp. 28-35 (1999).Examples include bacteria that produce toxins that requirefurin-mediated cleavage for into the cell, such as Pseudomonas exotoxinA, diphtheria toxin, and anthrax protective antigen. Certain human andanimal viruses contain glycoproteins that must be cleaved by host cellfurin before infectious particles can formed. Examples of such virusesinclude HIV and other retroviruses, fowl plague influenza virus, Semlikiforest virus, Newcastle disease virus, parainfluenza virus, measles,herpes, and Ebola. Furin thus represents a target for therapeuticattack. Although furin is required for the production of many importantcellular proteins, healthy cell lines exist that do not contain furin;suggesting that furin is not absolutely required for mammalian cellgrowth. It is likely that the temporary use of drugs affecting furin canpromote the antibacterial or antiviral activities of concurrently useddrugs acting by different mechanisms, thus selectively affectingpathogenesis rather than normal cellular activities.

[0088] Furin is also thought to be involved in the degradation ofextracellular matrix through its ability to activate precursors ofmatrix metalloproteinases (MMPs), in particular MMP-1. Since MMPexpression increases in many tumor cell types and has been implicated inmetastatic progression, inhibition of MMP activation by inhibiting furinmay result in the slowing of tumor progression. Thus furin may representa logical candidate for an anti-cancer drug.

[0089] Peptides that may be used in the present invention may have fromabout 4 to about 20 amino acids, preferably from about 6 to about 10amino acids. Not only are polyarginines useful in the present invention,but so are peptides comprising other basic amino acid residues, bothnaturally occurring, such as lysine and histidine, but also non-naturalor unusual basic amino acids, such as homoarginine, ornithine,diaminobutyric acid, and diaminopropionic acid. The amino acids may beD-form or L-form. Without wishing to be bound by this theory, it isbelieved that peptides having at least 4, preferably 6 to 9, consecutivebasic amino acid residues will have the greatest anti-furin activity.

[0090] As discussed above, it can be helpful to remove the acetyl andamide groups on the ends of the peptide to increase inhibitory effects,particularly the acetyl group.

[0091] As discussed above, peptides comprising D-amino acids are alsouseful in practicing this invention. Their inhibitory effects arecomparable to, though somewhat lower than those of theotherwise-identical peptides consisting of L-amino acids. However, sincetheir biological half-lives will in general be longer, D-amino acidpeptides may have advantages over L-amino acid peptides in practicingthis invention in vivo. D-nona-arginine, for example, is expected to bea useful anti-furin compound.

[0092] This method of treatment may be used in vertebrates generally,including human and non-human mammals, birds, fish, reptiles, andamphibians. Peptides in accordance with the present invention may beadministered to a patient by any suitable means, including oral,intravenous, parenteral, subcutaneous, intrapulmonary, and intranasaladministration. Oral administration may be best suited for D-formpeptides, since they are not broken down digestively. Oraladministration of D-form peptides may be enhanced by linking the peptideto a suitable carrier to facilitate uptake by the intestine, for examplevitamin B₁₂, following generally the B₁₂-conjugation technique of G.Russell-Jones et al., “Synthesis of LHRH Antagonists Suitable for OralAdministration via the Vitamin B₁₂ Uptake System,” Bioconjugate Chem.,vol. 6, pp. 34-42 (1995).

[0093] Parenteral infusions include intramuscular, intravenous,intraarterial, or intraperitoneal administration. The compound may alsobe administered transdermally, for example in the form of a slow-releasesubcutaneous implant, or orally in the form of capsules, powders, orgranules. It may also be administered by inhalation.

[0094] Pharmaceutically acceptable carrier preparations for parenteraladministration include sterile, aqueous or non-aqueous solutions,suspensions, and emulsions. Examples of non-aqueous solvents arepropylene glycol, polyethylene glycol, vegetable oils such as olive oil,and injectable organic esters such as ethyl oleate. Aqueous carriersinclude water, alcoholic/aqueous solutions, emulsions or suspensions,including saline and buffered media. Parenteral vehicles include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's, or fixed oils. The active therapeutic ingredient maybe mixed with excipients that are pharmaceutically acceptable and arecompatible with the active ingredient. Suitable excipients includewater, saline, dextrose, glycerol and ethanol, or combinations thereof.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers, such as those based on Ringer's dextrose, andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, inertgases, and the like.

[0095] The form may vary depending upon the route of administration. Forexample, compositions for injection may be provided in the form of anampule, each containing a unit dose amount, or in the form of acontainer containing multiple doses.

[0096] The compound may be formulated into therapeutic compositions aspharmaceutically acceptable salts. These salts include acid additionsalts formed with inorganic acids, for example hydrochloric orphosphoric acid, or organic acids such as acetic, oxalic, or tartaricacid, and the like. Salts also include those formed from inorganic basessuch as, for example, sodium, potassium, ammonium, calcium or ferrichydroxides, and organic bases such as isopropylamine, trimethylamine,histidine, procaine and the like. The compositions may be administeredintravenously, subcutaneously, intramuscularly, or (especially when inD-amino acid form and complexed with a carrier such as vitamin B₁₂)orally.

[0097] Controlled delivery may be achieved by admixing the activeingredient with appropriate macromolecules, for example, polyesters,polyamino acids, polyvinyl pyrrolidone, ethylenevinylacetate,methylcellulose, carboxymethylcellulose, prolamine sulfate, orlactide/glycolide copolymers. The rate of release of the active compoundmay be controlled by altering the concentration of the macromolecule.

[0098] Another method for controlling the duration of action comprisesincorporating the active compound into particles of a polymericsubstance such as a polyester, peptide, hydrogel, polylactide/glycolidecopolymer, or ethylenevinylacetate copolymers. Alternatively, an activecompound may be encapsulated in microcapsules prepared, for example, bycoacervation techniques or by interfacial polymerization, for example,by the use of hydroxymethylcellulose or gelatin-microcapsules orpoly(methylmethacrylate) microcapsules, respectively, or in a colloiddrug delivery system. Colloidal dispersion systems include macromoleculecomplexes, nanocapsules, microspheres, beads, and lipid-based systemsincluding oil-in-water emulsions, micelles, mixed micelles, andliposomes.

[0099] An “effective amount” of a peptide is an amount that inhibits theactivity of furin by a statistically significant degree; or thatinhibits the growth, metabolism, or reproduction of bacteria or virusesto a statistically significant degree; or that inhibits the growth ormetastasis of a tumor to a statistically significant degree; or thatablates the tumor to a statistically significant degree. “Statisticalsignificance” is determined as the P<0.05 level, or by such othermeasure of statistical significance as is commonly used in the art for aparticular type of experimental determination.

[0100] The complete disclosures of all references cited in thisspecification are hereby incorporated by reference. Also incorporated byreference is the full disclosure of the following paper, which is notprior art to this application: A. Cameron et al., “Polyarginines arepotent furin inhibitors,” J. Biol. Chem. vol. 275, pp. 36741-36749(2000). In the event of an otherwise irreconcilable conflict, however,the present specification shall control.

[0101] Abbreviations

[0102] Some of the abbreviations used in the specification follow:

[0103] Abz: o-aminobenzoyl

[0104] eddnp: ethylenediamine 2,4-dinitrophenyl

[0105] α1-PDX: α1-antitrypsin Portland

[0106] AMC: aminomethylcoumarin

[0107] D6R: D-polyarginine hexapeptide

[0108] TFA: trifluoroacetic acid

[0109] MCA: methylcoumarinamide

[0110] HEPES: N-[2-hydroxethyl]piperazine-N′-[2-ethanesulfonic acid]

[0111] MMPs: matrix metalloproteinases

[0112] MBP: major basic protein.

1 31 1 4 PRT Artificial Sequence misc_feature 2..3 Xaa in position twodenotes any amino acid. Xaa in position three denotes Lys or Arg. Thisis the reported consensus sequence for furin cleavage. 1 Arg Xaa Xaa Arg1 2 4 PRT Artificial Sequence misc_feature 2..3 Xaa in position twodenotes any amino acid. Xaa in position three denotes any amino acid.This is the reported minimum consensus sequence for furin cleavage. 2Arg Xaa Xaa Arg 1 3 7 PRT Meleagris gallopavo 3 Lys Pro Ala Cys Thr LeuGlu 1 5 4 7 PRT Artificial Sequence misc_feature Designed peptide. Thisis an engineered modification of SEQ ID NO 3, which is in turn derivedfrom Meleagris gallopavo. 4 Lys Pro Arg Cys Lys Arg Glu 1 5 5 7 PRT Homosapiens 5 Leu Glu Ala Ile Met Pro Ser 1 5 6 7 PRT Artificial Sequencemisc_feature Designed peptide. This is an engineered modification of SEQID NO 5, which is in turn derived from Homo sapiens. 6 Leu Glu Arg IleMet Arg Ser 1 5 7 10 PRT Artificial Sequence misc_feature Region of theprotease inhibitor alpha-2-macroglobulin. 7 Arg Val Gly Phe Tyr Glu SerAsp Val Met 1 5 10 8 10 PRT Artificial Sequence misc_feature Designedpeptide. This is an engineered modification of SEQ ID NO 7, which is inturn a region of alpha-2-macroglobulin. 8 Arg Val Gly Phe Tyr Glu SerAsp Val Met 1 5 10 9 10 PRT Homo sapiens 9 Val Val Arg Asn Ser Arg CysSer Arg Met 1 5 10 10 10 000 11 11 000 12 12 000 13 9 PRT ArtificialSequence misc_feature De novo designed peptide 13 Arg Arg Arg Arg ArgArg Arg Arg Arg 1 5 14 6 PRT Artificial Sequence misc_feature De novodesigned peptide 14 Arg Arg Arg Arg Arg Arg 1 5 15 6 PRT ArtificialSequence misc_feature De novo designed peptide 15 His His His His HisHis 1 5 16 6 PRT Artificial Sequence misc_feature De novo designedpeptide 16 His His Lys Arg Arg Arg 1 5 17 6 PRT Artificial Sequencemisc_feature De novo designed peptide 17 Met His Lys Arg Arg Arg 1 5 186 PRT Artificial Sequence misc_feature De novo designed peptide 18 LysHis Lys Arg Arg Arg 1 5 19 6 PRT Artificial Sequence misc_feature Denovo designed peptide 19 Arg His Lys Arg Arg Arg 1 5 20 6 PRT ArtificialSequence misc_feature De novo designed peptide 20 His Arg Lys Arg ArgArg 1 5 21 5 PRT Artificial Sequence misc_feature De novo designedpeptide 21 Met Arg Lys Arg Arg 1 5 22 6 PRT Artificial Sequencemisc_feature De novo designed peptide 22 Lys Arg Lys Arg Arg Arg 1 5 236 PRT Artificial Sequence misc_feature De novo designed peptide 23 ArgArg Lys Arg Arg Arg 1 5 24 6 PRT Artificial Sequence misc_feature Denovo designed peptide 24 Leu Leu Arg Val Lys Arg 1 5 25 4 PRT ArtificialSequence misc_feature De novo designed peptide 25 Arg Arg Arg Arg 1 26 5PRT Artificial Sequence misc_feature De novo designed peptide 26 Arg ArgArg Arg Arg 1 5 27 7 PRT Artificial Sequence misc_feature De novodesigned peptide 27 Arg Arg Arg Arg Arg Arg Arg 1 5 28 8 PRT ArtificialSequence misc_feature De novo designed peptide 28 Arg Arg Arg Arg ArgArg Arg Arg 1 5 29 10 PRT Artificial Sequence misc_feature Ten-residuesequence from furin propeptide. 29 Gln Gln Val Ala Lys Arg Arg Thr LysArg 1 5 10 30 10 PRT Artificial Sequence misc_feature Decapeptidefragment of the propeptide of PC7 30 Glu Gln Arg Leu Leu Lys Arg Ala LysArg 1 5 10 31 8 PRT Artificial Sequence misc_feature Hypothesized furincleavage fragment 31 Gln Gln Val Ala Lys Arg Arg Thr 1 5

We claim:
 1. A method for selectively inhibiting furin in a mammalianhost in need of furin inhibition, said method comprising administeringto the host an effective amount of a peptide having from four to twentyamino acid residues, wherein at least four consecutive amino acidresidues are basic.
 2. A method as recited in claim 1, wherein the hostis a human.
 3. A method as recited in claim 1, wherein the metabolism,growth, or reproduction of pathogenic bacteria in the host is reduced toa statistically significant degree by said inhibition of furin.
 4. Amethod as recited in claim 1, wherein the metabolism, growth, orreproduction of pathogenic viruses in the host is reduced to astatistically significant degree by said inhibition of furin.
 5. Amethod as recited in claim 1, wherein the growth or metastasis of atumor in the host is reduced to a statistically significant degree bysaid inhibition of furin, or wherein a tumor in the host is ablated to astatistically significant degree by said inhibition of furin.
 6. Amethod as recited in claim 1, wherein the peptide has from six to tenamino acid residues, and wherein at least six consecutive amino acidresidues are basic.
 7. A method as recited in claim 1, wherein thepeptide comprises L-form amino acid residues.
 8. A method as recited inclaim 1, wherein the peptide comprises D-form amino acid residues.
 9. Amethod as recited in claim 1, wherein the peptide comprisestetra-L-arginine (SEQ ID NO 25), penta-L-arginine (SEQ ID NO 26),hexa-L-arginine (SEQ ID NO 14), hepta-L-arginine (SEQ ID NO 27),octa-L-arginine (SEQ ID NO 28), or nona-L-arginine (SEQ ID NO 13).
 10. Amethod as recited in claim 1, wherein the peptide comprisestetra-D-arginine, penta-D-arginine, hexa-D-arginine, hepta-D-arginine,octa-D-arginine, or nona-D-arginine.
 11. A method as recited in claim 1,wherein the peptide lacks an N-terminal acetyl group, or wherein thepeptide lacks a C-terminal amide group, or wherein the peptide lacksboth an N-terminal acetyl group and a C-terminal amide group.
 12. Amethod as recited in claim 1, wherein the consecutive basic amino acidresidues are selected from the group consisting of arginine, histidine,and lysine.
 13. A method as recited in claim 1, wherein the consecutivebasic amino acid residues are selected from the group consisting ofarginine, histidine, lysine, homoarginine, ornithine, diaminobutyricacid, and diaminopropionic acid.